The present invention is directed to an automated system for microdissection of a sample such as chromosomes or other biological material, and in particular, it relates to a robotic assisted microdissection system and method that significantly reduces the time and skill needed for cellular and sub-cellular dissections.
Conventional surgical microdissection involves miniature tools performing cellular level dissection under a microscope. This can be an extremely tedious process. Miniature tools generally need to be fabricated in the laboratory by the researcher or technician using a micro-forge. The tools are then placed into the clamp of a micro-manipulator and moved about within the microscope""s field of view. The micro-manipulator scales the operator""s motion by some factor such as ten to one range. Even with the scaled movement of the micromanipulator, a great deal of practice and skill are needed to perform delicate operations such as chromosome dissection or in-vitro fertilization. Because microdissection is so physically demanding, it is a very low yield process (e.g., 4 per day), requiring frequent breaks from the work. In some situations large volumes of micro-dissected material are needed for significant (anywhere from 4 to 100) sampling to occur. For example, chromosome microdissection of a specific gene locus requires from 10 samples, up to 100 samples in order to gather enough material for subsequent processes. It can take up to a month for 100 microdissection samples to be taken. Manually positioning a micro-manipulator is a tedious process. A free standing micro-manipulator must be positioned by the operator sliding and bumping and tapping the base in an attempt to get the micro-tool point within the microscope""s field of view. If a new micro tool is used after each operation, the sliding, bumping and tapping process must be repeated each time.
Prior to the use of PCR in micro cloning, the construction of microdissection libraries had several drawbacks; e.g. the size of each such library was generally small; e.g., such a library contained at most only a few hundred clones of the microdissected material. However, for a dissected region of 10-20 mb, many more micro clones may be required to adequately generate libraries of the dissected region. Furthermore, since unstained and unbanded chromosome preparations were preferably used in microdissection, the identification of individual chromosomes and the chromosome bands was difficult and less accurate. Although suitable translocation stocks in mouse or human/rodent cell hybrids could be used, the prospect of a wider application to many other genomes was severely curtailed. Finally, the need to dissect several hundred chromosome segments for micro cloning would discourage investigators to use microdissection as a general method.
Focused UV laser microbeams have been used in conjunction with microdissection (e.g. Ponelies et al., 1989). In this procedure, all unwanted nuclei and chromosome regions on the slide are destroyed by a laser beam, leaving only the needed region intact which is then picked up and transferred to an Eppendorf tube for PCR.
Various other micromanipulating devices are known that utilize various means for manipulating material. For example, Parvin et al., U.S. Pat. No. 5,671,086 uses electrophoresis wherein electrodes are coupled to the stage of a microscope in an array whereby the electrode array allows for distinct manipulations of the electric field for accurate manipulations of an object. Kleindiek, U.S. Pat. No. 5,568,004 utilizes an electromechanical positioning device that is moved for course and fine adjustment by the operator when viewing a subject through a microscope, preferably using a piezo-tube connected to variable voltages to cause movements of a tip means. Higuchi et al., U.S. Pat. No. 5,225,750 disclose a micro-injection apparatus utilizing a micromovement device which relies upon a piezo electric/electro strictive element, which, in response to an electrical signal, causes a micromovement of a microsyringe.
Currently, the major obstacle to more widespread adoption of surgical microdissection technology is the extreme difficulty of the actual chromosome microdissection itself. This procedure takes a very experienced and skilled cytogeneticist. It requires continuous practice and a very significant amount of training. Surgical microdissection is also slow compared to possible automated procedures. It requires extreme care with regard to contamination. Finally, the preparation of the micro-tools requires for the procedure is also currently imprecise and requires an experienced individual. Only a few laboratories have the necessary resources and demand for the technique to have established it themselves.
Molecular analysis and high resolution physical mapping of specific genomic regions in any organism require large numbers of probes from the region of interest. A direct approach is to use microdissection techniques to physically remove the critical region followed by a micro cloning procedure to construct a region-specific library. These region-specific libraries are useful in a wide variety of studies, particularly for high resolution genome analysis and to facilitate positional cloning of important disease genes mapped to a specific region. Some examples of applications include: (i) isolation of large insert clones, like Pl, for contig assembly to cover a given genomic region; (ii) isolation of highly poly-morphic markers for linkage analysis; (iii) isolation of large numbers of single-copy micro clones as STSs for high density coverage of the region, (iv) isolation of region-specific CDNA clones as candidate genes for positional cloning, and (v) serving as chromosome painting probes to analyze complex chromosomal abnormalities.
The present invention is directed to an automated system to carry out microdissection for use by scientists who are not well equipped in genome analysis but are anxious to clone disease genes as their primary interest. This system is useful in laboratories for production of both microdissection painting probes for specific regions of the human or any other genome, including the genomes of plants, mice, and other model organisms currently of interest, e.g., aradopsis and Rugu rubripes (the Japanese pufferfish). The reliability and ease of operation of the automated system makes it widely available to laboratories that occasionally need microdissection technology but are not in a position to establish the technology as it currently stands due to the extremely demanding technical aspects of the conventional procedure.
Before the completion of the sequencing project for the entire human genome, there will be continuing needs for region-specific libraries and clones in specific regions for contig construction, refined physical mapping, linkage analysis, candidate gene isolation, etc. , all of which are essential to a successful cloning of a critical disease gene.
The automated system of the present invention greatly simplifies other microdissection procedures, for example microdissection of tumor specimens to separate the tumor cells from the normal surrounding tissue, and in vitro fertilization. Therefore, the automated system described herein has wide biomedical application. The present invention provides a device and method that makes it possible to construct microdissection libraries efficiently with high quality.
One aspect of the present invention relates to a method for removing select portions of a chromosome comprising calibrating a microdissection workstation by determining the position of a micro-tool using at least two CCD cameras and recording a zero position for the micro-tool. The micro-tool is then moved to a desired microscope objective (e.g., the crosshairs of the microscope) and the position of the micro-tool in such location is recorded. The micro-manipulator then releases the first micro-tool and selects a second micro-tool. Using the CCD cameras, the tip of the second micro-tool is determined and the differences in coordinate space between the zero position of the first micro-tool and the similar position of the second micro-tool are recorded. The second micro-tool is then moved beneath the microscope objective and is properly centered by adjusting the location of the second micro-tool using the differences between the zero position of the first micro-tool. A microscope slide having a chromosome in contact therewith is positioned beneath the microscope objective at a desired centered location (e.g., at the intersection of the microscope crosshairs). The second micro-tool is then brought into contact with the chromosome at the crosshair location in such a manner that a particular chromosome locus is removed from the chromosome. The removed chromosome portions are then deposited in a receptacle. The above-described method can be repeatedly performed using additional micro-tools to accumulate a desired amount of chromosome material.
As one will appreciate, the above-method is automated such that an operator need only expend the time and effort required to properly calibrate the system using a first micro-tool and thereafter rely upon the calibrated differences between such first measurements and measurements of subsequent micro-tools in order to achieve the desired repeated microdissection process.
The present invention also includes the automated microdissection apparatus utilized in the above-described method. In one embodiment, the workstation of the present invention uses an inverted microscope with a stage micrometer mounted onto a concentric substage, i.e., wherein the axis of rotation is about the optical axis of the microscope. Such substage capable of rotating a microscopic slide within the microscope""s field of view regardless of where the field of view is located on the microscopic slide. The rotating substage thus provides the ability to rotate a chromosome so as to be in alignment with a micromanipulator""s micro-tool. Motorization of the various axis of the microscopic stage allows automation of the stage to facilitate microdissection of particular chromosomes, regardless of the orientation of such chromosomes.
Yet another aspect of the present invention is directed to a micromanipulation device which comprises a microscope having a stage for placing a biological sample, a rotator for rotating the stage about the optical axes of the microscope, a first rectilinear device for moving the biological sample along a first axis on the stage, such rectilinear device being accurate in its movement to within a micron, and more preferably to within about {fraction (1/10)}th of a micron. A second rectilinear device is provided for moving the biological sample along a second axis on the stage, similarly being accurate in its movement to within a micron, and preferably, {fraction (1/10)}th of a micron. An end effector is provided capable of grasping a micro-tool. A user input device inputs data to achieve one or more of the following functions: activating rotation of the stage, activating the first rectilinear device for moving the biological sample along a first axis and activating the second rectilinear device for moving the biological sample along a second axis.
Using the present micromanipulation device, a biological sample is positioned in a desired orientation for manipulation, such manipulation achieved by contact of the biological material with the micro-tool. The micromanipulation device of the present invention may further include at least one spectral imaging component in addition to the microscope, such component providing information relating to the location of a micro-tool being used in the micromanipulation procedure. For example, when two imaging devices are positioned relative to the micro-tool, information is provided that locates the micro-tool in three-dimensional space. Other aspects of the present invention include a decontamination system for reducing the contamination of a biological sample, for example, one or more ultraviolet lamps. A further aspect relates to the use of a robotic controller for translating the input data into commands for activating one or more of the rotator and/or first or second rectilinear devices. The micro-tool can be directed through the use of a joystick, a mouse, hand wheels or a computer keyboard.
Other embodiments and aspects of the present invention will be obvious to one of skill in the art with the guidance provided by the attached figures and detailed description of preferred embodiments.